Protection against ischemia and reperfusion injury

ABSTRACT

A compound and method for using compound-D SEQ ID NO:1 to reduce injury associated with ischemia and reperfusion of mammalian organs such as the heart. The compound may be administered as part of a preconditioning strategy which reduces the extent of injury and improves organ function following cessation and restoration of blood flow. The compound may be used in preparation for planned ischemia or in a prophylactic manner in anticipation of further ischemic events.

[0001] This application claims the benefit of U.S. Application Ser. No.60/238,989, filed on Oct. 10, 2000.

FIELD OF THE INVENTION

[0002] The invention relates to compounds protective against ischemiaand reperfusion injury, particularly in the myocardium, and their use.

BACKGROUND

[0003] Tissues deprived of blood and oxygen undergo ischemic necrosis orinfarction with possible irreversible organ damage. In somecircumstances, however, such as during cardiac surgery, it is desirableto interrupt the normal myocardial contractions (cardioplegia) andactually induce ischemia. Such elective or obligatory ischemia occurs inthe presence of safeguards such as cardioplegia-induced cardiac arrestand hypothermia. While these safeguards provide considerable myocardialprotection, alteration of myocardial energetics (stunning) and poorpostoperative ventricular function still remain significant problems.

[0004] Once the flow of blood and oxygen is restored to the organ ortissue (reperfusion), the organ does not immediately return to thenormal preischemic state. Reperfused postischemic non-necroticmyocardium is poorly contractile and has reduced concentrations of highenergy nucleotides, depressed subcellular organelle function andmembrane damage that resolves only slowly. Although reperfusion restoresoxygen and reverses ischemia, repletion of high energy nucleotides suchas adenosine triphosphate (ATP) and reversal of ischemic membrane damageis slow, and contractile function may be profoundly depressed for a longperiod. Just minutes of ischemia causes loss of myocardial systolic wallthickening for hours. Longer periods of reversible ischemia may depresscontractility for days. Studies confirm that, despite restoration ofmyocardial flow and a quick recovery of myocardial oxygen consumption(MVO₂) following ischemia, there is only very slow recovery ofmyocardial contractile function. The problems are exacerbated in highrisk patients, such as those with poor preoperative ventricularfunction, recent myocardial infarction or left ventricular hypertrophy.These same problems also occur during organ storage for cardiactransplant, under which there are time constraints due to the limits ofmyocardial preservation.

[0005] Postischemic dysfunction may be due to a variety of factors.Oxygen free radicals may play a role, as generation of free radicals instunned myocardium has been demonstrated and free radical scavengershave been shown to attenuate contractile dysfunction. Impairedintracellular calcium handling and calcium overload during earlyreperfusion may contribute to postischemic dysfunction; while calciuminfusions enhance contractility in both normal and postischemicmyocardium, ischemia as short as a few minutes produces an impairment insarcoplasmic reticulum calcium transport and a shift of the calciumATPase activity. Postischemic myocardium is also associated with reducedconcentrations of myocardial high-energy phosphates and adeninenucleotides, as obligatory reduction in myocardial ATP content duringischemia occurs as myocytes utilize ATP for maintenance of cellularintegrity. Since ATP is essential for myocardial contraction andrelaxation, ATP depletion may have detrimental effects upon postischemicmyocardial functional recovery.

[0006] The high volume of cardiac-related surgeries, both elective andemergency procedures and including cardiac transplants, lead to theabove-described problems. Thus, methods and agents to provide protectionagainst myocardial ischemia and to avoid post ischemic dysfunction areneeded.

SUMMARY OF THE INVENTION

[0007] The invention is directed to an agent and a method of using theagent to reduce the injury associated with ischemia and reperfusion oforgans such as the heart. The compound isTyr-D-Leu-Phe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH₂SEQ ID NO:1, hereinafter referred to as compound-D. Administration ofcompound-D SEQ ID NO:1, particularly prior to an ischemic event, reducestissue necrosis and preserves organ function.

[0008] In one embodiment, a method of protecting against ischemia andreperfusion injury in a mammal is disclosed. An effective concentrationof compound-D SEQ ID NO:1 is administered to the mammal in apharmaceutically acceptable formulation prior to the onset of ischemia,for example, 24 hours prior to ischemia. In other embodiments,compound-D SEQ ID NO:1 is administered substantially concurrent with theonset of ischemia, during an ischemic episode, or post-ischemia. Theformulation may be administered parenterally at a concentration in therange of about 1-20 mg/kg of body weight.

[0009] The invention is also directed to a method to prevent damage toan isolated organ, for example, a heart for transplant. The isolatedorgan is exposed to a preservative solution containing an effectiveamount of compound-D SEQ ID NO:1. The concentration of compound-D SEQ IDNO:1 in the preservative solution for a heart is about 100 μM.

[0010] The invention is additionally directed to a method for reducingeffects of an ischemic episode in a mammal by administering an effectiveconcentration of compound-D SEQ ID NO:1 in a pharmaceutically acceptablecarrier. Administration is prior to or substantially concurrently withthe onset of ischemia, or one hour post cerebral ischemia.

[0011] The invention is further directed to a composition that protectsa mammalian organ from injury. The composition contains compound-D SEQID NO:1. Compound-D may be naturally occurring or may be synthesized.

[0012] The invention is also directed to an organ preservative solutionthat contains compound-D at a concentration effective to protect theorgan, such as a heart, from ischemic injury.

[0013] These and other advantages of the invention will be apparent inlight of the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a histogram showing myocardial infarction size incontrol and treated animals.

[0015]FIG. 2 is a histogram showing post-ischemic release of creatinekinase in control and treated animals.

[0016]FIG. 3 is a histogram showing post-ischemic release of troponin Iin control and treated animals.

[0017]FIG. 4 is a graph showing end diastolic pressure in the leftventricle of control and treated animals.

[0018]FIG. 5 is a graph showing functional recovery in control andtreated animals.

[0019]FIG. 6 is a graph showing coronary flow in control and treatedanimals.

[0020]FIG. 7 is a histogram showing the effect of naltrexone on infarctsize in animals pretreated with compound-D SEQ ID NO:1.

[0021]FIG. 8 is a histogram showing the effect of glibenclamide on vasdeferens contractility in animals treated with 1,000 nM compound-D SEQID NO:1.

[0022]FIG. 9 is a histogram showing the effect of glibenclamide on vasdeferens contractility in animals treated with 2,000 nM compound-D SEQID NO: 1.

[0023]FIG. 10 is a histogram showing the effect of compound-D SEQ IDNO:1 on cell metabolic activity.

[0024]FIG. 11 is a histogram showing the effect of compound-D SEQ IDNO:1 on cell viability.

[0025]FIG. 12 is a graph showing cell viability and metabolic activitywith increasing concentrations of compound-D SEQ ID NO:1.

[0026]FIG. 13 is a histogram showing the effect of compound-D SEQ IDNO:1 on cells exposed to oxidative damage.

DETAILED DESCRIPTION

[0027] The invention is directed to a compound that has a salutaryeffect on cardiac function following ischemia, and methods of using thecompound. The compound may be administered directly to an individual,and is particularly effective when administered 24 h prior to the onsetof ischemia. This may occur, for example, prior to scheduled cardiacsurgery. The compound may also be included in a preservative solutionfor an isolated organ, such as a heart or liver being maintained viablefor transplant.

[0028] The compound is a peptide having the sequenceTyr-D-Leu-Phe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH₂SEQ ID NO: 1, hereinafter referred to compound-D. The peptide may beproduced by a number of methods, such as using an automated peptidesynthesizer, through recombinant molecular techniques, or isolated froma naturally occurring source, as is known to one skiIled in the art.Compound-D SEQ ID NO:1 has a molecular weight of 1,902 daltons.Compound-D SEQ ID NO:1 is insoluble in water or saline, but may besolubilized by adding 100 μM of a solution comprised of ethanol,propylene glycol, and 1 N NaOH in a 1:1:1 ratio, with sterilephysiological saline then used to obtain the appropriate concentration.The initial alkaline pH is adjusted to 7.4 with 1 N HCl.

[0029] Compound-D SEQ ID NO:1 that has been solubilized may beadministered by parenteral means, for example, by intravenous injection.In one embodiment, administration of compound-D SEQ ID NO:1 is at thetime of induced ischemia, fut may also be added during or even after anischemic event. For administration into a mammal, a dose of about 1-20milligrams per kilogram (mg/kg) is useful. For administration into atissue or organ preservation solution, a concentration of about 100 μMis useful.

[0030] Compound-D SEQ ID NO:1 may be administered directly into amammal, either alone or in combination with other substances.Alternatively, it may be added as a component of a solution used tomaintain the viability of isolated organs, such as an additive tocardioplegia and other organ preservation solutions. In one embodiment,compound-D SEQ ID NO:1 is coadministered as an adjuvant with othercompounds or strategies that are designed to protect organs fromischemia. As an example, compound-D SEQ ID NO:1 may be administered withagents that affect nitric oxide (NO) synthase, such as argininehydrochloride. Arginine hydrochloride is known to prevent the decline incardiac function following an ischemic episode.

[0031] The following description demonstrates use and efficacy ofcompound-D SEQ ID NO:1 in a variety of systems.

[0032] Perfused Heart

[0033] Compound-D SEQ ID NO:1, at a dose of 2 mg/kg and solubilized asdescribed above, was administered by tail vein injection into ratsweighing between about 350-400 g (number of animals (n)=6). Control rats(n=6) were injected in the same manner with an equal volume of 0.9%NaCl. After 24 h, the hearts from both treated and control animals wereexcised and perfused in a modified Langendorff perfusion apparatus at37° C. using oxygenated Krebs-Henseleit buffer as the perfusate, asknown to one skiIled in the art. Coronary perfusion pressure wasmaintained at 700 mm Hg by regulating coronary flow. All hearts werepaced at 5.5 Hz (300 beats per minute, bpm) except during ischemia. Asaline-fiIled balloon was inserted in the left ventricle to measuredeveloped pressure (DP) and end-diastolic pressure (EDP) in mm Hg. After15 min equilibration, both groups were subjected to 20 min zero-flowglobal ischemia, and then were reperfused for 120 min.

[0034] Left ventricles were isolated and divided into three segmentsalong their short axis, stained with triphenyltetrazolium chloride, andstored in formalin. Infarct size was measured on digitized images andexpressed as a percentage of myocardium. Data were expressed as meanplus or minus standard error of the mean (±SEM) and were analyzed usinga paired Student's t-Test. Confidence limits were established at 95%.

[0035] The results are shown in FIG. 1, which is a histogram of theinfarct size in hearts from both animals receiving only saline (control,solid bar) and in animals treated with compound-D SEQ ID NO:1 at 2.0mg/kg (treated, open bar) 24 h prior to 20 min ischemia and 120 minreperfusion. Pretreatment of animals with compound-D SEQ ID NO:1significantly decreased the percent of infarct size (p<0.05). Controlrats had a mean infarct size of 27±5%, while rats pretreated withcompound-D had a reduced mean infarct size of 12±3%.

[0036] Compound-D SEQ ID NO:1 pretreatment also significantly decreasedtotal creatine kinase (CK) and the cardiac form of troponin I (cTn-1)values following 20 min ischemia, as shown in FIGS. 2 and3,respectively. CK is an enzyme that is normally found in muscles suchas the heart (that is, in myocytes), but is released during severemuscle trauma such as occurs in a myocardial infarction. Thus,measurement of CK is an indicator of the degree of muscle damage ortrauma. Similarly, cTn-I is a protein associated specifically withcardiac muscle, and an increase in cTn-1 indicates myocardial damage.

[0037]FIG. 2 is a histogram showing CK released during reperfusion ofisolated rat hearts after 20 min ischemia in rats treated 24 h prior toischemia with 2.0 mg/kg compound-D SEQ ID NO:1. The solid bars representcontrol animals (n=6) and the open bars represent treated animals (n=6).FIG. 3 is a histogram showing cTn-I released during reperfusion ofisolated rat hearts after 20 min ischemia in rats treated 24 h prior toischemia with 2.0 mg/kg compound-D SEQ ID NO:1. The solid bars representcontrol animals (n=6) and the open bars represent treated animals (n=6).At each time point during reperfusion where samples were coIlected forCK and cTn-1 analysis (1, 15, 30, 60, 90 and 120 min reperfusion), CKlevels in control rats were higher than CK levels in treated rats, andTn-1 levels in control rats were higher than Tn-1 levels in treatedrats. This indicated that pretreatment with compound-D SEQ ID NO:1decreased the damage to the myocardium, as compared to animals that didnot receive compound-D SEQ ID NO:1.

[0038] Compound-D SEQ ID NO:1 pretreatment also resulted in improvedpostischemic ventricular function. FIG. 4 is a graph of end diastolicpressure (EDP) in mm/Hg in the left ventricle during reperfusion ofisolated rat hearts after 20 min ischemia in rats treated 24 h prior toischemia with 2.0 mg/kg compound-D SEQ ID NO:1. Open squares are fromtreated animals, and solid circles are from control animals. FIG. 5 is agraph showing percent of functional recovery during reperfusion ofisolated rat hearts after 20 min ischemia in rats treated 24 h prior toischemia with 2.0 mg/kg compound-D SEQ ID NO:1. Open squares are fromtreated animals, and solid circles are from control animals. Differencesin recovery of developed pressure (DP) of compound-D SEQ ID NO:1 treatedhearts remained statistically significant to 90 min following initiationof reperfusion, as shown in FIG. 4, and in end diastolic pressure to 20min following initiation of reperfusion, as shown in FIG. 5. Leftventricular functional recovery (% recovery of baseline preischemicdeveloped pressure during reperfusion) was markedly improved in treatedanimals (open squares) compared to control animals (solid circles) to 90min of reperfusion. As shown in FIG. 6, coronary flow was notsignificantly different between control animals (solid circles) andcompound-D SEQ ID NO:1 treated animals (open squares).

[0039] These results show that in a normoxic, isolated perfused ratheart preparation, administration of compound-D can confercardioprotection when administered 24 h prior to planned ischemia. Thesalutary effects on the post-ischemic myocardium include reduced infarctsize, decreased release of both creatine kinase and cardiospecifictroponin I, and improved ventricular performance.

[0040]FIG. 7 shows the effect of naltrexone, a universal opioidantagonist, on infarct size. Groups of rats were pretreated 24 h priorto ischemia with either compound-D SEQ ID NO:1 at a dose of 2.0 mg/kg(n=6), naltrexone at a dose of 3 mg/kg (n=6), or a combination ofcompound-D SEQ ID NO:1 at a dose of 2.0 mg/kg and naltrexone at a doseof 3 mg/kg (n=6). Control rats (n=6) received vehicle (1:1:1 solution of1N NaOH:propyleneglycol:ethanol, readjusted to 7.4 with HCl) only. After24 h pretreatment, isolated rat hearts were subjected to 20 min ischemiafollowed by 120 min reperfusion.

[0041] As shown in FIG. 7, these data indicate that naltrexoneadministered in combination with compound-D SEQ ID NO:1 did not blockthe cardioprotective effects of compound-D SEQ ID NO:1. In fact, thedata support a synergistic cardioprotective effect from administrationof a combination of compound-D SEQ ID NO:1 and naltrexone.Administration of compound-D SEQ ID NO:1 reduced infarct size from about27% in the control group (solid bar) to about 13% in the compound-D SEQID NO:1 treated group (open bar) (p=0.05). The combined preischemicadministration of compound-D SEQ ID NO:1 and naltrexone (downwardhatched bar) reduced infarct size to about 10% when compared to thecontrol group (p=0.05). Additionally, these data demonstrate thatcompound-D SEQ ID NO:1 does not operate via delta opioid receptors,since naltrexone, an opioid receptor antagonist, did not preventcompound-D SEQ ID NO:1 activity in reducing infarct size (about 14.5%versus about 13%, respectively).

[0042] Mouse Vas Deferens

[0043] Utilizing a mouse vas deferens (m.v.d.) assay, compound-D SEQ IDNO:1, it was demonstrated that opioid receptor binding requiresactivation and/or opening of K_(ATP) channels. Glibenclamide, a K_(ATP)channel blocker, blocked the inhibition of contraction of mouse vasdeferens induced by compound-D SEQ ID NO:1. With reference to FIG. 8,the effect of administration of 1000 nM compound-D SEQ ID NO:1 alone(open bar) versus 1000 nM compound-D SEQ ID NO:1 with 6000 nMglibenclamide (hatched bar) on contractility of mouse vas deferens(m.v.d.) (n=6) is shown. Glibenclamide restored m.v.d. contractionswhich compound-D SEQ ID NO:1 had suppressed. FIG. 8 shows that 1000 nMcompound-D SEQ ID NO:1 suppressed contractility to above 100%. FIG. 8also shows that differences between control contractions and 1000 nMcompound-D SEQ ID NO:1, as well as control versus 6000 nM glibenclamide,and 1000 nM compound-D SEQ ID NO:1 versus 6000 nM glibenclamide arestatistically significant. FIG. 9 shows the effect of administration of2000 nM compound-D alone (open bars) versus 2000 nM compound-D SEQ IDNO:1 with 6000 nM glibenclamide (hatched bar) on contractility of m.v.d.(n=2).

[0044] These results demonstrate that the effect of compound-D SEQ IDNO:1 requires opening and/or activation of K_(ATP) channels sinceglibenclamide, a potassium channel blocker, effectively blocked thesmooth muscle inhibition of contraction induced by compound-D SEQ IDNO:1 in the m.v.d. assay. The possibility therefore exists thatadministration of compound-D SEQ ID NO:1 and interventions directlytargeted at opening K_(ATP) channels by drugs such as nicorandil ordiazoxide, or anesthetics such as isoflurane, could be effectiveapproaches for pharmacologically duplicating both short term and longterm (second window) ischemic preconditioning. Thus, preinfusions ofcompound-D SEQ ID NO:1 may provide extended protection to the ischemicmyocardium in a variety of clinical scenarios.

[0045] Compound-D, SEQ ID NO:1 has also been shown to be effective inreducing ischemic effects when administered 1 h after an episode ofinduced cerebral ischemia. A solution of 100 μL of either vehicle alone(control) or compound-D (treated) at a concentration of 1 mg/ml wasadministered either 1 h before cerebral ischemia or 1 h after cerebralischemia (n=6 in each group). In animals receiving compound-D beforeischemia, there were no significant differences in infarct volume(control 91±10 mm³ versus treated 89±11 mm³) or neurological score(control group 1.93±0.29 versus treated group 1.95±0.33). However, whencompound-D was administered 1 h post ischemia, there were significantdifference in both the infarct volume (control group 90±13 mm³ versustreated group 56±9 mm³) and the neurological score (control group1.91±0.27 versus treated group 1.42±0.28) in rats receiving compound-DSEQ ID NO:1 versus the control group. The mean arterial blood pressure,pO₂,pCO₂, pH and cerebral blood flow showed no significant differencesin control versus treated groups when compound-D SEQ ID NO:1 wasadministered either pre- or post-cerebral ischemia (data not shown).These results suggest a therapeutic role for compound-D SEQ ID NO:1 inmitigating the effects of a cerebral ischemic episode that is inprogress or that has occurred.

[0046] Cultured Cell Lines

[0047] The rat pheochromocytoma cell line, PC12(ATCC; CRL-1721) wascultured in growth medium, RPMI 1640 medium supplemented with 10 mMHEPES, 1 mM sodium pyruvate, 2 mM L-glutamine (GIBCO BRL), 10% horseserum (HS; Sigma), and 5% fetal bovine serum (FBS; JRH). Ten to twelvedays prior to assay, PC12cells were induced with 50 ng/ml recombinanthuman beta nerve growth factor (NGF; R&D Systems) to the neuronalphenotype on collagen I coated tissue culture dishes. Two days prior toassay, cells were plated on collagen IV coated 48-well tissue cultureplates without NGF at 3-5×10⁵ cells per well.

[0048] For hypoxic/ischemic conditions, the medium was changed to eitherDulbecco's Modified Eagle Medium (DMEM; GIBCO BRL) without glucose, orDMEM with 5 mM glucose. Both were supplemented with 10 mM HEPEScontaining compound-D SEQ ID NO:1 at various concentrations. Cells wereplaced in an environmental chamber (Plas-Labs) maintained at 37° C. andperfused with either a gas mixture of 85% nitrogen, 10% hydrogen and 5%carbon dioxide to induce anoxic conditions, or a mixture of air and 5%carbon dioxide to induce normoxic conditions. After four hours, plateswere removed from the chambers and medium was changed to growth mediumwith compound-D SEQ ID NO:1 at appropriate concentrations. Cells werereturned to the air-5% CO₂ incubator until the next day.

[0049] Alamar Blue (AccuMed International Companies), containing afluorometric/colorimetric oxidation/reduction indicator, was added tothe assay medium in an amount equal to 10% of the assay volume. Cellswere returned to the incubator for two to four hours. Fluorescence ofeach well was measured at an excitation wavelength of 530 nm and anemission wavelength of 580 nm in a CytoFluor Multi-well Plate Reader(PerSeptive Biosystems). The average fluorescence readings from threewells were used as a relative measurement of the metabolic activity ofthe cells.

[0050] Viability was assessed in the same wells using a crystal violetstaining technique. Cells were washed twice with phosphate bufferedsaline (PBS). Cells were lysed and stained with 0.5% crystal violet in20% methanol. Fifty microliters of this solution was left in the wellsfor five minutes. The plates were then washed under slow running waterabout five times or until the water ran clear. Excess water was removedby inverting and tapping the plates on an absorbent surface. Stainedcells were solublized with at least 200 μl/well of a solution of 0.1%sodium citrate in 50% ethanol. After ten minutes, 50 μl of solublizedstain was transferred to a 96-well microtiter plate and read at anabsorbance of 570 nm and subtracted from reference wavelength of 650 nm.The average readings from three wells were used as a relativemeasurement of the viability of the cells.

[0051] As shown in FIG. 10, cell metabolic activity as assessed byAlamar Blue uptake by PC12 cells was increased with increasingconcentrations of compound-D SEQ ID NO:1.

[0052] As shown in FIG. 11, cell viability as assessed by crystal violetuptake of PC12 cells was increased with increasing concentrations ofcompound-D SEQ ID NO:1.

[0053] As shown in FIG. 12, NGF differentiated cells under ischemicconditions and treated with increasing concentrations of compound-D SEQID NO:1 (bottom line) showed increasing viability, with the highestconcentration of compound-D SEQ ID NO:1 showing cell viability slightlyexceeding the positive control. Also as shown in FIG. 12, NGFdifferentiated cells under suboptimal metabolic conditions and treatedwith increasing concentrations of compound-D SEQ ID NO:1 (top line)showed increasing cell viability.

[0054] Oxidative damage has been suggested to play a critical role in anumber of cardiovascular diseases including atherosclerosis, fetalarrhythmia, aortic stenosis, cardiac hypertrophy, or in valvulardiseases where prolonged cardiac ischemia is a factor. Duringreperfusion, reactive oxygen species including hydrogen peroxide (H₂O₂)and hydroxyl radicals are found in the myocardium. These oxidants can bederived from various sources such as activated neutrophils, xanthineoxidase, mitochondrial respiration, and arachidonic acid metabolism. Theresulting oxidative damage has been shown to cause cell death in manyexperimental systems. (Chen, Q. M., Bahl, J. J., Archives ofBiochemistry and Biophysics, Vol. 373, No. 1, 242-248, Jan. 1, 2000).

[0055] The rat myoblast cell line H9C2 was used to measure myocardialcell necrosis. H9C2 cells were plated onto 96 well plates and grown toconfluency in DMEM +10% FBS. Plates were washed one time with assaybuffer consisting of phenol red free DMEM +2.5% FBS supplemented withnonessential amino acids, sodium pyruvate and HEPES buffer. All testreagents were made using the assay buffer.

[0056] Compound-D SEQ ID NO:1 was diluted in assay buffer to 2×finalconcentrations, ranging from 20 nM to 0.2 nM. H₂O₂ stock (30%) wasdiluted to 2×final concentration (0.2 mM) in assay buffer.

[0057] Immediately following the wash step, 50 μl 2×compound-D SEQ IDNO:1 at varying concentrations or assay buffer only (control) was addedto quadruplicate wells of a 96 well plate. Cells were incubated at 37°C. under 5% CO₂ for thirty minutes.

[0058] Following the pre-incubation, 50 μl of 2×(0.2 mM) H₂O₂ was addedto all wells except the controls (media only, no treatment). The finalH₂O₂ concentration was 0.1 mM. The plate was again incubated at 37° C.for four hours.

[0059] The cell supernatants were immediately assayed for lactatedehydrogenase (LDH) release using the CytoTox 96 Non-RadioactiveCytotoxicity Assay (Promega, catalogue #G 1780), following themanufacturer's assay procedures. LDH is a stable cytosolic enzyme thatis released upon cell lysis. It is used to measure either cell mediatedcytoxicity or cytotoxicity mediated by chemicals or other agents, andprovides a sensitive marker of cell damage and necrosis. Briefly, 50 μlsupernatant samples from each well were transferred to another 96 wellplate. Assay media only and total cell lysates were included ascontrols. Substrate mix (50 μl ) was added to all wells and the platewas covered and incubated in the dark for thirty minutes at roomtemperature. Stop solution (50 μl) was added to each well and theabsorbance at 490 nM was recorded. Assay media only absorbance valueswere subtracted to correct for any LDH activity that may be present inthe assay buffer.

[0060] As shown in FIG. 13, cell necrosis due to oxidative damage wasdecreased in the presence of compound- D SEQ ID NO:1.

[0061] In summary, a compound that is administered to a mammal to reducethe injury associated with ischemia and reperfusion of organs such asthe heart is disclosed. The compound isTyr-D-Leu-Phe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH₂SEQ ID NO:1. Administration prior to an ischemic event reduces tissuenecrosis and preserves organ function, as measured by standardphysiological indices of organ function. The compound may beadministered in a time frame from 24 h up to 15 min prior to plannedischemia, or even during an ischemic period.

[0062] It should be understood that the embodiments of the presentinvention shown and described in the specification are only preferredembodiments of the inventors who are skiIled in the art and thus are notlimiting in any way. For example, compound-D SEQ ID NO:1 may be used asa protective agent for organs other than the heart. Organs isolated fortransplant such as kidney, liver, lung, cornea, etc. may have enhancedviability after exposure to compound-D SEQ ID NO:1. Similarly,compound-D SEQ ID NO:1 may be administered to individuals in whichischemia to organs other than the heart has occurred or will occur.Therefore various changes, modifications or alterations to theseembodiments may be made or resorted to without departing from the spiritof the invention and the scope of the following claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211>LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: XYZ <220> FEATURE: <221>NAME/KEY: PEPTIDE <222> LOCATION: (0)...(0) <221> NAME/KEY: MOD_RES<222> LOCATION: (1)...(0) <223> OTHER INFORMATION: Xaa = D-Leu <400>SEQUENCE: 1 Tyr Xaa Phe Ala Asp Val Ala Ser Thr Ile Gly Asp Phe Phe HisSer 1 5 10 15 Ile

What is claimed is:
 1. A method of protecting against ischemia andreperfusion injury in a mammal comprising administering an effectiveamount of compound-D SEQ ID NO:1 to the mammal in a pharmaceuticallyacceptable formulation.
 2. The method of claim 1 wherein administrationis prior to onset of ischemia.
 3. The method of claim 2 whereinadministration is about 24 hours prior to onset of ischemia.
 4. Themethod of claim 1 wherein compound-D SEQ ID NO:1 is administered as asolution.
 5. The method of claim 1 wherein compound-D SEQ ID NO:1 isadministered parenterally.
 6. The method of claim 1 wherein compound-DSEQ ID NO:1 is administered at a concentration in the range of about 2mg/kg of body weight of the mammal.
 7. The method of claim 1 whereincompound-D SEQ ID NO:1 is administered to protect against myocardialischemia.
 8. The method of claim 1 wherein the formulation furtherincludes a compound having an anti-ischemic effect.
 9. The method ofclaim 8 wherein the compound affects nitric oxide synthase.
 10. Themethod of claim 9 wherein the compound is arginine hydrochloride.
 11. Amethod to preserve an isolated organ comprising exposing the organ to asolution comprising a preservative and an effective amount ofcompound-D.
 12. The method of claim 11 wherein the organ is selectedfrom the group consisting of a heart and a liver.
 13. The method ofclaim 11 wherein compound-D SEQ ID NO:1 is at a concentration of about100 μM in the solution.
 14. A method for reducing effects of ischemia ina mammal comprising administering an effective amount of compound-D SEQID NO:1 in a pharmaceutically acceptable carrier to the mammal.
 15. Themethod of claim 14 wherein compound-D SEQ ID NO:1 is administered priorto ischemia.
 16. The method of claim 14 wherein compound-D SEQ ID NO:1is administered substantially concurrently with onset of ischemia. 17.The method of claim 14 wherein compound-D SEQ ID NO:1 is administeredafter one hour of cerebral ischemia.
 18. A composition comprisingcompound-D SEQ ID NO:1.
 19. The composition of claim 18 whereincompound-D SEQ ID NO:1 is obtained by in vitro synthesis.
 20. Thecomposition of claim 18 wherein compound-D SEQ ID NO:1 is isolated froma natural source.
 21. A solution to preserve viability of an isolatedorgan comprising compound-D SEQ ID NO:1 at a concentration effective toprotect the organ from ischemic injury.
 22. The solution of claim 20wherein the organ is selected from the group consisting of a heart and aliver.